Phytase (myo-inositol hexakisphosphate phosphorylase, EC 3.1.3.8) catalyses the hydrolysis of the phosphor-ester bonds of phytate (myo-inositol hexakisphosphate). Over the last decade and a half, fungal and bacterial phytases have been successfully used as an additive in the diet of monogastric animals to improve bioavailability of phytate phosphate and other minerals. In order to measure the enzymatic activity of this animal feed additive, Engelen and colleagues published a “simple and rapid” method for measuring phytase activity (Engelen A J, et al., Simple and rapid determination of phytase activity. J AOAC Int'l. 77(3):760-64 (1994)). The assay conditions (37.0° and pH 5.5) prescribed by these authors were the optimal reaction conditions for Aspergillus niger phytase (Natuphos™), the only phytase available at that time in the market. An official version of this basic method was published in 1996 in the fourth edition of the Food Chemicals Codex (FCC). In this document, a phytase (fytase) unit (FTU) was defined as “the amount of enzyme that liberates inorganic phosphate at 1 μmol/min from sodium phytate 0.0051 mol/L at 37.0° C. at pH 5.50 under the conditions of the test.”
A modified version of the FCC IV phytase method for the purpose of measuring phytase activity in animal feed was published by Engelen et al. (Determination of phytase activity in feed by a colorimetric enzymatic method: collaborative interlaboratory study. J. AOAC Int'l. 84:629-33 (2001)). This method was also published as AOAC Official Method 2000.12 Phytase Activity in Feed and is generally referred to as the “AOAC method.” It is essentially the same as the FCC method but includes additional steps for grinding and extracting feed samples prior to enzyme assay.
The AOAC method for measuring phytase in feed, however, lacks high precision and robust reproducibility, especially for feed samples from feed batches dosed pre-pelleting with granular or particulate dry formulations of phytases and also for Escherichia coli (E. coli) derived phytases. Therefore, there was a need to adapt the AOAC in-feed phytase method for phytases added pre-pelleting and more specifically for E. coli derived phytases added pre-pelleting.
Traditionally, enzymes were sprayed onto the feed pellets after the pelleting step because the enzymes were not thermostable and could not survive the high temperatures used in the pelleting process, which includes steam conditioning. When the enzyme was sprayed onto the cooled feed pellets, the enzyme was distributed more evenly and only on the surface of the pellet. More recently, enzymes are being added to the mash or dry mixed feed pre-pelleting as concentrated granular or particulate dry formulations, where the enzyme is applied to a carrier or embedded in a carrier or bulking agent. The dry product particles may also be wax-coated, for example, for improved heat resistance. A key advantage to this type of concentrated dry formulation is that it can be added to feed using existing dry microingredient handling systems standard at most feed mills. A major drawback of dry concentrates added pre-pelleting, however, is that the enzyme activity is delivered not as a uniform spray but in a concentrated particulate form and at an extremely low inclusion rate. This creates new challenges relating to uniform product distribution throughout the feed batch (mixing) that has implications for both enzyme delivery to animals (i.e., uniform dosing) as well as the reproducibility of analytical methods (i.e., enzyme assay reproducibility from sub-sample to sub-sample).
New thermostable enzymes are being used in animal feed (Garrett et al., Applied and Environmental Microbiology 70(5): 3041-3046 (2004)). The new thermostable enzymes can be added pre-pelleting since they can withstand the high temperatures used in the steam conditioning and pelleting process. Using traditional in-feed analytical methods did not allow for reliable or reproducible measurements of the enzyme activity of the feed samples. The enzyme activities measured were highly variable between assays of sub-samples of the same feed samples. Possible factors contributing to this variability include a) non-uniform distribution of dry product particles, especially in samples of mash feed batches due to settling of product in stored samples, b) non-representative batch samples and sub-samples resulting from insufficient sample size, c) inefficient extraction of enzyme from granulated dry product particles in which the enzyme is embedded in the carrier or bulking agent or trapped in the feed matrix as a result for example of starch gelatinization, and d) low signal-to-noise ratio in activity assay due to sub-optimal assay conditions.
Dry formulated enzymes are typically added to feed at very low inclusion rates, resulting in extremely low concentrations of enzyme in the complex mixture of the feed. For example, the recommended dose of QUANTUM Phytase (an E. coli derived phytase) 2500D for broiler is 500 FTU/kg feed. This dose is achieved at an inclusion rate of 200 grams of 2500D per metric ton of feed, which corresponds to a 5000-fold dilution of the product in feed. Therefore, a highly sensitive assay is required for accurate measurement of phytase activity in feed. For maximum assay sensitivity it is critical that the conditions of the assay are set at the optimal pH and temperature for the enzyme. As mentioned above, the assay conditions for phytase activity measurement described in the AQAC method (Engelen, A. J., et al. 2001) and which define the standard phytase unit or FTU are optimal for Aspergillus niger phytase. However, these conditions are not optimal for phytases derived from other sources, such as E. coli, and when used compromise assay sensitivity and reliability.
Improving the signal-to-noise ratio for phytase activity measurements increases assay accuracy but does not however reduce the observed large variation in assay results across both replicate feed batch samples and across replicate sub-samples of batch samples. Factors contributing to assay variability across samples and sub-samples were mentioned above and include heterogeneous distribution of the phytase enzyme in the sample and inefficient or non-quantitative extraction of the enzyme, especially from steam conditioned and pelleted feed. These observations demonstrate the need for consistent sample processing as well as an efficient procedure for extracting phytase activity from animal feed samples.
Therefore, there was an unmet need for a reproducible and reliable measurement of enzyme activity in feed samples when the enzyme is added pre-pelleting or added as a granule. The present invention has significant improvements to the traditional in-feed enzyme assay making it possible to obtain reproducible, reliable measurements of enzyme activity in replicate samples taken from animal feed batches. The improvements of the present invention are using a larger feed batch sample size, grinding the feed batch sample to a smaller particle size, and using an alkaline pH extraction buffer.